First in 1997, and later re-emerging in 2003, highly pathogenic avian influenza A virus, subtype H5N1, has spread from wild bird reservoirs to domestic bird flocks. As a result, cross-transmission has been confirmed in people living or working in close contact with infected birds. H5N1 virus infection is associated with a high mortality rate (>60%) in humans and the rapid evolution of the virus suggests that it could potentially develop into a new, and possibly severe, pandemic influenza virus. To-date, conventional inactivated and live-attenuated vaccine strategies offers the best protection against influenza virus infection; however, poor immunogenicity and weaker efficacy have been observed against H5N1 viruses. It was hypothesized that experimental adenovirus-based vaccines based on human adenovirus serotype 5 (AdHu5) or porcine adenovirus serotype 3 (PAV3) can offer protection against a broad range of avian influenza, subtype H5N1, viruses. Ad vaccine vectors are highly immunogenic and have demonstrated protective efficacy against several disease models. However, natural immunity against AdHu5 can interfere with vector efficacy. The nonhuman PAV3 was not neutralized by pooled human serum from 10,000-60,000 individuals and offers a promising alternative to AdHu5-based vectors. Systematic antigen screening using DNA vaccines identified the hemagglutinin (HA) glycoprotein as the most immunogenic H5N1 antigen. HA was then inserted directly into PAV3 or AdHu5. Comparable immune responses were observed between both vectors but, interestingly, the PAV3-based vaccine generated stronger T-cell responses and better rapid protection 8 days following immunization. Additionally, better long-term protection 1 year following vaccination was observed with the PAV3-HA vaccine. The co-administration of multiple H5N1 antigens was also screened to improve protection against divergent H5N1 challenge. Combinations of DNA vaccines expressing (HA+NA) and (HA+NP) offered the best promise for enhancing protection against homologous and heterologous H5N1 challenges, respectively. However, addition of three or more antigens reduced overall protection possibly by antigen dilution, competition, or interference. Co-administration of PAV3 or AdHu5 vectors expressing both the HA and NP antigens reduced protection against homologous and heterologous H5N1 virus challenges. For all combination vaccines, T-cell responses were strong against HA but significantly decreased against additional antigens in each combination vaccine. Overall, the experimental porcine-based Ad-based vaccine offered better protection than the H5N1 conventional vaccine against a broad range of different H5N1 viruses. Understanding of the relationship between immune parameters and protection will be critical in future improvement of adenovirus-based and other vaccines against avian influenza H5N1.

First in 1997, and later re-emerging in 2003, highly pathogenic avian influenza A virus, subtype H5N1, has spread from wild bird reservoirs to domestic bird flocks. As a result, cross-transmission has been confirmed in people living or working in close contact with infected birds. H5N1 virus infection is associated with a high mortality rate (>60%) in humans and the rapid evolution of the virus suggests that it could potentially develop into a new, and possibly severe, pandemic influenza virus. To-date, conventional inactivated and live-attenuated vaccine strategies offers the best protection against influenza virus infection; however, poor immunogenicity and weaker efficacy have been observed against H5N1 viruses. It was hypothesized that experimental adenovirus-based vaccines based on human adenovirus serotype 5 (AdHu5) or porcine adenovirus serotype 3 (PAV3) can offer protection against a broad range of avian influenza, subtype H5N1, viruses. Ad vaccine vectors are highly immunogenic and have demonstrated protective efficacy against several disease models. However, natural immunity against AdHu5 can interfere with vector efficacy. The nonhuman PAV3 was not neutralized by pooled human serum from 10,000-60,000 individuals and offers a promising alternative to AdHu5-based vectors. Systematic antigen screening using DNA vaccines identified the hemagglutinin (HA) glycoprotein as the most immunogenic H5N1 antigen. HA was then inserted directly into PAV3 or AdHu5. Comparable immune responses were observed between both vectors but, interestingly, the PAV3-based vaccine generated stronger T-cell responses and better rapid protection 8 days following immunization. Additionally, better long-term protection 1 year following vaccination was observed with the PAV3-HA vaccine. The co-administration of multiple H5N1 antigens was also screened to improve protection against divergent H5N1 challenge. Combinations of DNA vaccines expressing (HA+NA) and (HA+NP) offered the best promise for enhancing protection against homologous and heterologous H5N1 challenges, respectively. However, addition of three or more antigens reduced overall protection possibly by antigen dilution, competition, or interference. Co-administration of PAV3 or AdHu5 vectors expressing both the HA and NP antigens reduced protection against homologous and heterologous H5N1 virus challenges. For all combination vaccines, T-cell responses were strong against HA but significantly decreased against additional antigens in each combination vaccine. Overall, the experimental porcine-based Ad-based vaccine offered better protection than the H5N1 conventional vaccine against a broad range of different H5N1 viruses. Understanding of the relationship between immune parameters and protection will be critical in future improvement of adenovirus-based and other vaccines against avian influenza H5N1.

No description available.

Three potential driver genes were identified by use of retroviral mutagenesis in the newly developed iMyc model of B cell malignancy. To do so, iMyc mice, which bear an inserted copy of c-Myc in the IgH locus, were treated with MOL4070LTR to favor the development of B cell malignancies. After tumor development, B cell origin tumors were identified by use of immunohistochemistry and selected for downstream analysis. Three genes were chosen as potential driver genes and validated in mouse or human disease as involved in disease or directly in malignancy. These genes are CD82, IRAK2 and DNMT3a.

Tuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M. tuberculosis, therefore a long treatment time is required for stable cure without relapse. Poor patient compliance increases the emergence of drug resistant strains, posing yet another challenge for the eradication of TB. There is dire need for novel compounds targeting not only drug-resistant, but also dormant bacteria so as to effectively eliminate drug-resistant strains and also shorten treatment time. This requires compounds with novel modes of action and novel drug screening approaches which focus on dormant M. tuberculosis. In the current work a method was optimized which induces the dormant phenotype of M. tuberculosis in vitro allowing large scale screening of compounds against these tolerant bacteria. The high chemical diversity of marine natural products was explored to increase the chances of finding novel compounds with novel mechanisms of action. Additionally, gold-complexed scaffolds were examined for their putative ability to inhibit topoisomerase 1, which is a highly conserved and essential protein of mycobacteria, not currently targeted in classical treatment regimens. Several marine natural products were identified with selective bactericidal activity against dormant bacteria, emphasizing the powerful tool that was developed for drug screening. Moreover, the gold-complexes were also bactericidal against not only replicating and dormant bacilli, but also mycobacteria resistant to front-line TB drugs. Compounds characterized in this study represent a promising starting point for the development of novel TB therapeutics and discovery of new conditionally essential pathways of dormant bacteria.

No description available.

Pacific salmon migrate long distances to spawn as part of their life cycle. During this journey from sea to their natal stream, they undergo major endocrine, physiological and immune changes. Cortisol, the primary stress hormone, gradually increases during the journey. Persistent high cortisol levels have deleterious health effects, including suppression of the antibody response. However, pathogens encountered during their journey may stimulate antibody responses to overcome the infection. My main research question focuses on how salmonids balance the immunosuppressive effects of high cortisol levels with activation of the antibody response. A recent field study from our lab showed a transient increase in abundance of B cells during the spawning run which is suggestive of activation of the immune system during this journey. However, our field study had too many confounding variables. In this study, we investigated the activation of the antibody response under conditions of elevated levels of cortisol in rainbow trout under laboratory-controlled conditions. We looked at the effects of a) cortisol alone, b) fish pathogen Flavobacterium psycrophilum (Fp) alone and c) combined cortisol and Fp challenge on the gene expression of immunoglobulins IgM and IgT using qPCR. We have found that cortisol suppresses the IgM response in the spleens of Fp-susceptible line but not in Fp-resistant line of Rainbow trout. No significant effects on B cell development where observed in the anterior kidney. Taken together, our data suggest that the antibody response in Fp-resistant rainbow trout is less sensitive to increased cortisol levels compared to Fp-susceptible fish, confirming our hypothesis that Fp-resistant fish have in some way evolved to manage stress more successfully.

Human immunodeficiency virus (HIV) infects and destroys lymphocytes, leading to acquired immunodeficiency syndrome. Homosexual men were impacted disproportionately in the early years of the pandemic, while women are now the majority of those infected, making development of anti-HIV treatments and preventatives effective in the female reproductive tract (FRT) imperative. Here, we investigated HIV prevention in women through 1) discovery of antivirals effective in the FRT; and 2) determining a mechanism by which bacterial vaginosis (BV), a disorder of the FRT in which protective Lactobacillus are replaced by BV-associated bacteria (BVAB), increases HIV transmission. We identified a small molecule active against HIV from an extensive compound library and used its structure for an in silico screening, identifying a novel class of HIV inhibitors, Avirulins. Three were active in the low micromolar range and exhibited HIV-1 reverse transcriptase inhibition. Avirulins were not cytotoxic to FRT epithelial cells and maintained activity in human cervicovaginal fluid (CVF). With continued development, Avirulins could serve as additions to antiretroviral therapies or preventatives for the FRT. Next, we hypothesized that BV increases HIV transmission though disruption of the FRT epithelium at the endocervical monolayer, as most FRT lymphocytes reside below the epithelium. We determined that matrix metalloproteinases (MMPs) were secreted by endocervical epithelium in response to BVAB and could depolarize endocervical cell layers. When HIV infected lymphocytes were cocultured with endocervical cell layers, treatment with conditioned media from endocervical cells cocultured with BVAB increased HIV transmigration. Treatment with MMP inhibitors reduced this effect. We demonstrated that CVF from women with BV had greater MMP activity, and presence of certain MMP isotypes in CVF correlated with increased HIV transmigration through the endocervical epithelium. These results propose endocervical disruption by MMPs as a mechanism for BV-induced HIV transmission and suggest the potential of MMP inhibitors as HIV preventatives.

No description available.

Mycobacterium tuberculosis (M.tb) is the causative agent of pulmonary tuberculosis (TB). Over 25% of the world’s population is estimated to be infected with tuberculosis, yielding over 10 million new cases and over 1.5 million deaths in 2017 alone. This is all despite the near universal implementation of bacille Calmette-Guérin (BCG) vaccination across TB endemic areas. BCG is capable of preventing childhood disseminated forms of disease but fails to induce potent immunity within the lung. We expect this to in part play a role in the lack of protection against pulmonary TB. Our lab has developed a human adenovirus serotype 5 vaccine expressing the M.tb antigen Ag85A (AdHu5Ag85A). When delivered directly to the respiratory mucosa (RM), AdHu5Ag85A has proven to be safe and immunogenic, generating both CD4 and CD8 T cell responses within the lung. We have found that RM AdHu5Ag85A vaccination also generates a long-lasting population of memory macrophages in the airway and lung, that are primed to better control early M.tb infection. Importantly, this was a vaccination route-dependent mechanism. Memory macrophages express a trained innate immune phenotype as they express high levels of MHC II, are highly glycolytic, and produce more IL-12 upon re-stimulation. Importantly, utilization of an in vivo T cell depletion approach allowed us to show that these memory macrophages are capable of limiting early M.tb infection independent of T cells. Our findings indicate that RM vaccination may be advantageous to parenteral routes by not only drawing antigen specific T cells into immunologically restricted lung mucosa but also generating a memory macrophage population within the lung. Induction of memory macrophages within the airway helps overcome early innate immune suppression by M.tb. / Thesis / Master of Health Sciences (MSc) / Tuberculosis (TB) is a pulmonary disease responsible for 10 million new clinical cases and more than 1 million deaths annually. Over one quarter of the world is believed to be infected with TB. This is despite the near-universal administration of bacille Calmette-Guérin, a preventative TB vaccine, and an effective, but lengthy antibiotic treatment. With antibiotic resistance on the rise, developing a protective vaccine against TB is more important than ever. Tuberculosis has confounded researchers for decades and has thus escaped development of a more effective vaccine. One of the ways to improve a TB vaccine would be to inhale it, to have local effects at the main site of TB infection in the lung. We found that by aerosolizing our vaccine, we can impact local immunity within the lung in a way that has never before been described, opening new avenues for TB research.